Z.S. Gardner et al.
Bioorganic & Medicinal Chemistry Letters 45 (2021) 128136
delocalize their positive charge across a large surface area and thus do
not require any specific transporters for mitochondrial trans-
location19–22. In addition, TPP+ cations have been shown to localize
inside the mitochondria matrix compared to the cytoplasm, in some
cases there is 1,000-fold concentration differences. These TPP+ cations
have also been extensively studied as mitochondrial targeting anticancer
Table 1
Cell proliferation inhibition (IC50) values of Baylis-Hillman aryl phosphonium
bromide salts (MTT assay). IC50 values reported as average values ± SEM from
three separate experiments.
and other agents19–22
.
–
The Baylis-Hillman (BH) reaction is a well-established C C bond
forming reaction in organic chemistry to synthesize functionalized allyl
alcohols and amines under simple reaction conditions23–26. The allyl
alcohols can be readily converted into corresponding allyl bromides
under standard bromination conditions. These bromides are known to
undergo reactions with a wide variety of nucleophiles in SN2 or SN2′
fashion to provide further functionalized synthetic intermediates23–26
.
We recently reported a protocol by converting BH bromides to their
corresponding aryl acetates by reacting them with arylcarboxylic acids.
Several of these aryl acetates showed significant in vitro cell proliferation
inhibition properties against various cancer cells. We envisioned that the
aryl acetates acted as a leaving group for cellular nucleophiles to provide
the cytotoxicity27. Our long-standing interest in the development of
novel small molecule anticancer agents27–32 has prompted us to syn-
thesize and evaluate novel functionalized phosphonium salts derived
from BH bromides and aryl phosphines. We hypothesized that these
novel phosphonium salts could be targeted to mitochondria to develop
them as anticancer agents. Based on the reactivity of the alkox-
ycarbonylallyl ester group to cellular nucleophiles, we also envisioned
that these agents would interact with mitochondrial nucleophiles to
potentially disrupt mitochondrial integrity and energetics leading to
inhibition of cancer cell proliferation.
phosphonium derivative synthesized from tris(2-furyl) phosphine and
BH bromide didn’t show any significant activity even at 100 µM con-
centration indicating that this compound is not sufficiently lipophilic to
diffuse into mitochondrial matrix.
To further understand the SAR of these compounds, the alkoxy ester
group was modified by replacing the methyl group with metabolically
more stable tBu and N,N-dimethyl groups. The required BH bromides
were synthesized via literature known protocols28,33,34. The reaction of
these bromides with triphenyl phosphine in acetonitrile, followed by
recrystallization in ether gave the corresponding phosphonium bro-
mides 5–7 in good yields (Scheme 1). Cell proliferation inhibition
studies of 5–7 showed an increase in potency with tBu substitution
compared to methyl group. The enhanced carbon content and lipophilic
nature of the tBu group may allow for better tissue diffusion and bio-
logical potency. Moreover, the tBu ester is metabolically more stable to
enzymatic hydrolysis than the methyl ester and is a preferred option
over the methyl ester for in vivo studies. Surprisingly, the metabolically
stable N,N-dimethyl amide derivative 7 did not show any significant
activity even at high concentrations of 100 µM in all the tested cell lines
(Table 1). This could be explained due to the lesser nucleophile
accepting capability of alkyl amides compared to corresponding esters
resulting in the reduced biological potency; further illustrating that the
ester moiety is necessary for reactivity with intracellular nucleophiles
and subsequent biological activity. Encouraged by the higher activity of
the tBu ester in 5, the tBu bromide was reacted with tris(4-
methoxyphenyl)phosphine and recrystallized in ether to obtain the
corresponding phosphonium bromide 6. This compound showed even
further enhancement in cell proliferation inhibition from the combina-
tion of enhanced lipophilicity and better cationic stabilization due to
methoxy groups (Table 1).
To synthesize the aryl phosphonium salts, initially, we utilized BH
reaction derived methyl 2-(bromomethyl)acrylate as a model substrate.
The BH product was synthesized in two steps by the reaction of para-
formaldehyde with methyl acrylate in the presence of DABCO followed
by the bromination of resulting alcohol. The bromide was reacted with
various mono-substituted triaryl phosphines including triphenyl, 4-
methyl, 4-methoxy and 4-fluoro triaryl, tris(2-furyl) and cyclohexyl
diphenyl phosphines. In all the cases, 2-(alkoxycarbonyl)-allyl-TPP de-
rivatives 1–5 were obtained in good yields after purification by recrys-
tallization (Scheme 1).
The synthesized phosphonium derivatives 1–5 were evaluated for
their in vitro cell proliferation inhibition properties against several solid
tumor cell lines by utilizing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) cell viability assay. Human breast cancer
MCF7, human triple-negative breast cancer MDA-MB-231, human
pancreatic cancer MiaPaCa-2, human colorectal adenocarcinoma WiDr,
murine metastatic breast cancer 4T1, and murine breast cancer 67NR
cell lines were utilized in this study. From these studies, we found that
the tris(4-methylphenyl) phosphonium 2 and tris(4-methoxyphenyl)
phosphonium 4 were more biologically potent on all the tested cells
lines with IC50 values ranging from 1 to 10 µM (Table 1). The electron
donating character of the tolyl and methoxy groups may stabilize the
cation and enhance the ability of compounds 2 and 4 to diffuse down
into the mitochondrial matrix, leading to higher mitochondrial accu-
mulation and potency. A cyclohexyldiphenyl phosphonium synthesized
from the corresponding phosphine and BH bromide showed relatively
lower activity (9–67 µM) compared its triaryl derivatives. A tris(2-furyl)
We further carried out SAR studies on the β-substitution of BH bro-
mides to explore the effect of β-substitution on biological activity. To
synthesize the required β-substituted BH bromides, a variety of electron
withdrawing and donating aromatic aldehydes were used. These bro-
mides were obtained under standard BH reaction and subsequent
bromination conditions. Since tris(4-methylphenyl) phosphine provided
higher cell proliferation properties, phosphonium derivatives 8–16 were
synthesized using β-substituted BH bromides with this phosphine
Scheme 2. Synthesis of phosphonium salts with tris(4-methox-
Scheme 1. Synthesis of 2-carbonyl-allyl phosphonium bromides.
yphenyl)phosphine.
2